This invention pertains to a human leucocyte type of 12-LO and its role in the pathogenesis of several major disease states.
AA=Arachidonic acid
AII=Angiotensin II
EGF=Epidermal Growth Factor
FN=Fibronectin
GAPDH=Glyceraldehyde-3-phosphate dehydrogenase
GF=Growth Factor
HAEC=Human Aortic Endothelial Cells
HETE=Hydroxyeicosatetraenoic Acid
12 HETE=12-Hydroxyeicosatetraenoic Acid
HG=High Glucose
hl 12-LO=Human Leukocyte 12-Lipoxygenase
hl 15-LO=Human Leukocyte 15-Lipoxygenase
HODE=Hydroxyoctadecadienoic acid
12-HPETE=12-Hydroperoxyeicosatetraenoic Acid
HSMC=Human Aortic Smooth Muscle Cells
HPLC=High Pressure Liquid Chromatography
IL-1 =Interleukin-1
LDL=Low Density Lipoprotein
LO=Lipoxygenase
12-LO=12-Lipoxygenase
15-LO=15-Lipoxygenase
MAPK=Mitogen Activated Kinase
mmLDL=Minimally Modified Low Density Lipoprotein
MO=Monocytes
NIDDM=Non-insulin Dependent Diabetes Mellitus
NG=Normal Glucose
PDGF=Platelet Derived Growth Factor
PKC=Protein Kinase C
pl 12-LO=Human platelet 12-Lipoxygenase
PVSMC=Porcine Vascular Smooth Muscle Cells
RT-PCR=Reverse Transcriptase Polymerase Chain Reaction
SMC=Smooth Muscle Cells
TGFB=Transforming Growth Factor Beta
TNF=Tumor Necrosis Factor 1
VSMC=Vascular Smooth Muscle Cells
The three mammalian lipoxygenases are named according to the carbon position (1, 2 or 3) at which they oxygenate arachidonic acid (4). There is increasing evidence that certain LO enzymes are involved in the pathogenesis and acceleration of atherosclerosis by inducing oxidation of LDL to its atherogenic form (5,6) and increasing the growth or migration of smooth muscle cells (1, 7-9). In addition, evidence suggests that a 12-LO protein plays a role in mediating angiotensin II (AII) induced vascular and adrenal actions (10-12). Recent studies indicate that at least two forms of 12-LO exist, i.e., pl 12-LO cloned from human erythroleukemia cells (2,13) and a porcine leukocyte 12-LO which has been isolated and cloned from porcine mononuclear cells, pituitary (14) and bovine tracheal cells (15).
Applicants have demonstrated the presence of a leukocyte type of 12-LO in human adrenal glomerulose cells (3). The human 15-LO has been purified from human and rabbit reticulocytes (16,17). The human platelet and porcine leukocyte type 12-LO share 65% amino acid homology (13). However, porcine leukocyte type 12-LO is highly homologous to human 15-LO (86%) (14). Recently, it has been shown that 15-LO is expressed in macrophages of human atherosclerotic lesions but not in unstimulated monocytes (18).
This application describes evidence for the presence of a human leucocyte type of 12-LO enzyme (hl 12-LO) and its role in the pathogenesis of several major disease states or processes, including atherosclerosis, breast cancer, autoimmune and inflammatory disease, diabetic vascular and kidney disease and insulin resistance. There are several features of this unique enzyme that can link several seemingly diverse conditions.
1. hl 12-LO Can utilize arachidonic and linoleic acid as fatty acid substrates generating hydroperoxides and other lipid mediators which can activate important signal transduction pathways commonly associated with these disorders. These mediators include (a) kinases such as specific isoforms of protein kinase C and mitogen activated kineses (MAPK), (b) transcription factors such as NFkB and oncogenes which have clearly been implicated in inflammatory and autoimmune conditions, atherosclerosis, cancer growth and metastasis.
2. Activation of the hl 12-LO enzyme can itself generate superoxide anions which can lead to the propagation of free radical processes which could accelerate the oxidative modification of lipids and proteins. These processes are involved in the pathogenesis of inflammatory, neoplastic and atherogenic conditions.
3. The hl 12-LO enzyme is strategically located. Evidence is presented showing the presence of the leucocyte type 12-LO in human monocytes, aortic vascular smooth muscle and endothelial cells, cardiac myocytes, skeletal muscle, the kidney and breast cancer cells and beta cells of pancreatic islets. These sites of activity of this enzyme allow a tissue specific role in leading to pathologic states. For instance, in the beta cells of the pancreatic islets, activation of 12-LO activity by inflammatory cytokines (e.g., IL-1) could explain the selective dysfunction and destruction of the beta or insulin producing cells of the pancreas. Furthermore, activation or increased expression of the 12-LO pathway by glucose in the beta cells could explain the dysfunctional secretion of insulin in the common form of adult diabetes (non-insulin dependent diabetes).
4. Factors increasing 12-LO expression and activity are linked to inflammatory, atherosclerotic, renal and neoplastic disease.
The factors demonstrated to increase the activity and expression of 12-LO include, (a) inflammatory cytokines associated with autoimmune disease (Type I diabetes) atherosclerosis and neoplastic growth such as interleukin-1xcex2 (IL-1), (b) growth factors such as platelet derived growth factor (PDGF) and angiotensin II (AII) which have been implicated in accelerated vascular and kidney disease, and (c) hyperglycemia which has been linked to the microvascular (eye, kidney and nerve) and microvascular (heart attack, stroke and peripheral vascular disease) complications of both type I or type II diabetes.
5. Applicants have found that glucose which accounts for much of the acquired insulin resistance in diabetes increases 12-LO activity and expression in all tissues tested. Therefore, 12-LO activation could provide a common link between glucose-induced oxidative stress and development of end-organ dysfunction or damage.
Pursuant to this invention, blockade of the hl 12-LO expression or enzyme activation provides novel treatments to prevent these disease states.
Applicants have now evaluated the precise type of LO present in unstimulated human aortic smooth muscle cells (HSMC), endothelial cells (HAEC) and monocytes (MO). Furthermore, since AII can increase the expression of 12-LO in human adrenal cells, applicants have also evaluated the effects of AII on 12-LO regulation in HSMC. Finally, applicants determined whether immunohistochemical analysis of atherosclerotic lesions demonstrates the presence of a leukocyte type of 12-LO. The results show that a 12-LO similar to that found in human adrenal glomerulose is expressed in the normal HSMC, HAEC and MO. Furthermore, this 12-LO is markedly upregulated by AII in HSMC and is present in human atherosclerotic lesions.